Matrix proteins are inefficiently imported into Arabidopsis peroxisomes lacking the receptor-docking peroxin PEX14

Mutations in peroxisome biogenesis proteins (peroxins) can lead to developmental deficiencies in various eukaryotes. PEX14 and PEX13 are peroxins involved in docking cargo-receptor complexes at the peroxisomal membrane, thus aiding in the transport of the cargo into the peroxisomal matrix. Genetic screens have revealed numerous Arabidopsis thaliana peroxins acting in peroxisomal matrix protein import; the viable alleles isolated through these screens are generally partial loss-of-function alleles, whereas null mutations that disrupt delivery of matrix proteins to peroxisomes can confer embryonic lethality. In this study, we used forward and reverse genetics in Arabidopsis to isolate four pex14 alleles. We found that all four alleles conferred reduced PEX14 mRNA levels and displayed physiological and molecular defects suggesting reduced but not abolished peroxisomal matrix protein import. The least severe pex14 allele, pex14-3, accumulated low levels of a C-terminally truncated PEX14 product that retained partial function. Surprisingly, even the severe pex14-2 allele, which lacked detectable PEX14 mRNA and PEX14 protein, was viable, fertile, and displayed residual peroxisome matrix protein import. As pex14 plants matured, import improved. Together, our data indicate that PEX14 facilitates, but is not essential for peroxisomal matrix protein import in plants.     

The peroxisome biogenesis factors pex4p, pex22p, pex1p, and pex6p act in the terminal steps of peroxisomal matrix protein import

Peroxisomes are independent organelles found in virtually all eukaryotic cells. Genetic studies have identified more than 20 PEX genes that are required for peroxisome biogenesis. The role of most PEX gene products, peroxins, remains to be determined, but a variety of studies have established that Pex5p binds the type 1 peroxisomal targeting signal and is the import receptor for most newly synthesized peroxisomal matrix proteins. The steady-state abundance of Pex5p is unaffected in most pex mutants of the yeast Pichia pastoris but is severely reduced in pex4 and pex22 mutants and moderately reduced in pex1 and pex6 mutants. We used these subphenotypes to determine the epistatic relationships among several groups of pex mutants. Our results demonstrate that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, the Pex5p docking factors Pex13p and Pex14p, the matrix protein import factors Pex8p, Pex10p, and Pex12p, and two other peroxins, Pex2p and Pex17p. Pex22p and the interacting AAA ATPases Pex1p and Pex6p were also found to act after Pex10p. Furthermore, Pex1p and Pex6p were found to act upstream of Pex4p and Pex22p. These results suggest that Pex1p, Pex4p, Pex6p, and Pex22p act late in peroxisomal matrix protein import, after matrix protein translocation. This hypothesis is supported by the phenotypes of the corresponding mutant strains. As has been shown previously for P. pastoris pex1, pex6, and pex22 mutant cells, we show here that pex4Delta mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.     

Meiotic development initiation in the fungus Podospora anserina requires the peroxisome receptor export machinery

Peroxisomes are versatile organelles essential for diverse developmental processes. One such process is the meiotic development of Podospora anserina. In this fungus, absence of the docking peroxin PEX13, the RING-finger complex peroxins, or the PTS2 co-receptor PEX20 blocks sexual development before meiocyte formation. However, this defect is not seen in the absence of the receptors PEX5 and PEX7, or of the docking peroxins PEX14 and PEX14/17. Here we describe the function of the remaining uncharacterized P. anserina peroxins predictably involved in peroxisome matrix protein import. We show that PEX8, as well as the peroxins potentially mediating receptor monoubiquitination (PEX4 and PEX22) and membrane dislocation (PEX1, PEX6 and PEX26) are indeed implicated in peroxisome matrix protein import in this fungus. However, we observed that elimination of PEX4 and PEX22 affects to different extent the import of distinct PEX5 cargoes, suggesting differential ubiquitination-complex requirements for the import of distinct proteins. In addition, we found that elimination of PEX1, PEX6 or PEX26 results in loss of peroxisomes, suggesting that these peroxins restrain peroxisome removal in specific physiological conditions. Finally, we demonstrate that all analyzed peroxins are required for meiocyte formation, and that PEX20 function in this process depends on its potential monoubiquitination target cysteine. Our results suggest that meiotic induction relies on a peroxisome import pathway, which is not dependent on PEX5 or PEX7 but that is driven by an additional cycling receptor. These findings uncover a collection of peroxins implicated in modulating peroxisome activity to facilitate a critical developmental cell fate decision.     

Identification and functional characterization of Arabidopsis PEROXIN4 and the interacting protein PEROXIN22

Peroxins are genetically defined as proteins necessary for peroxisome biogenesis. By screening for reduced response to indole-3-butyric acid, which is metabolized to active auxin in peroxisomes, we isolated an Arabidopsis thaliana peroxin4 (pex4) mutant. This mutant displays sucrose-dependent seedling development and reduced lateral root production, characteristics of plant peroxisome malfunction. We used yeast two-hybrid analysis to determine that PEX4, an apparent ubiquitin-conjugating enzyme, interacts with a previously unidentified Arabidopsis protein, PEX22. A pex4 pex22 double mutant enhanced pex4 defects, confirming that PEX22 is a peroxin. Expression of both Arabidopsis genes together complemented yeast pex4 or pex22 mutant defects, whereas expression of either gene individually failed to rescue the corresponding yeast mutant. Therefore, it is likely that the Arabidopsis proteins can function similarly to the yeast PEX4-PEX22 complex, with PEX4 ubiquitinating substrates and PEX22 tethering PEX4 to the peroxisome. However, the severe sucrose dependence of the pex4 pex22 mutant is not accompanied by correspondingly strong defects in peroxisomal matrix protein import, suggesting that this peroxin pair may have novel plant targets in addition to those important in fungi. Isocitrate lyase is stabilized in pex4 pex22, indicating that PEX4 and PEX22 may be important during the remodeling of peroxisome matrix contents as glyoxysomes transition to leaf peroxisomes.     

The importomer peroxins are differentially required for peroxisome assembly and meiotic development in Podospora anserina: insights into a new peroxisome import pathway

Peroxisome biogenesis relies on two known peroxisome matrix protein import pathways that are mediated by the receptors PEX5 and PEX7. These pathways converge at the importomer, a peroxisome‐membrane complex that is required for protein translocation into peroxisomes and consists of docking and RING–finger subcomplexes. In the fungus Podospora anserina, the RING–finger peroxins are crucial for meiocyte formation, while PEX5, PEX7 or the docking peroxin PEX14 are not. Here we show that PEX14 and the PEX14‐related protein PEX14/17 are differentially involved in peroxisome import during development. PEX14/17 activity does not compensate for loss of PEX14 function, and elimination of both proteins has no effect on meiocyte differentiation. In contrast, the docking peroxin PEX13, and the peroxins implicated in peroxisome membrane biogenesis PEX3 and PEX19, are required for meiocyte formation. Remarkably, the PTS2 coreceptor PEX20 is also essential for meiocyte differentiation and this function does not require PEX5 or PEX7. This finding suggests that PEX20 can mediate the import receptor activity of specific peroxisome matrix proteins. Our results suggest a new pathway for peroxisome import, which relies on PEX20 as import receptor and which seems critically required for specific developmental processes, like meiocyte differentiation in P. anserina.     

The Arabidopsis peroxisomal targeting signal type 2 receptor PEX7 is necessary for peroxisome function and dependent on PEX5

Plant peroxisomal proteins catalyze key metabolic reactions. Several peroxisome biogenesis PEROXIN (PEX) genes encode proteins acting in the import of targeted proteins necessary for these processes into the peroxisomal matrix. Most peroxisomal matrix proteins bear characterized Peroxisomal Targeting Signals (PTS1 or PTS2), which are bound by the receptors PEX5 or PEX7, respectively, for import into peroxisomes. Here we describe the isolation and characterization of an Arabidopsis peroxin mutant, pex7-1, which displays peroxisome-defective phenotypes including reduced PTS2 protein import. We also demonstrate that the pex5-1 PTS1 receptor mutant, which contains a lesion in a domain conserved among PEX7-binding proteins from various organisms, is defective not in PTS1 protein import, but rather in PTS2 protein import. Combining these mutations in a pex7-1 pex5-1 double mutant abolishes detectable PTS2 protein import and yields seedlings that are entirely sucrose-dependent for establishment, suggesting a severe block in peroxisomal fatty acid beta-oxidation. Adult pex7-1 pex5-1 plants have reduced stature and bear abnormally shaped seeds, few of which are viable. The pex7-1 pex5-1 seedlings that germinate have dramatically fewer lateral roots and often display fused cotyledons, phenotypes associated with reduced auxin response. Thus PTS2-directed peroxisomal import is necessary for normal embryonic development, seedling establishment, and vegetative growth.     

A viable Arabidopsis pex13 missense allele confers severe peroxisomal defects and decreases PEX5 association with peroxisomes

Peroxisomes are organelles that catabolize fatty acids and compartmentalize other oxidative metabolic processes in eukaryotes. Using a forward-genetic screen designed to recover severe peroxisome-defective mutants, we isolated a viable allele of the peroxisome biogenesis gene PEX13 with striking peroxisomal defects. The pex13-4 mutant requires an exogenous source of fixed carbon for pre-photosynthetic development and is resistant to the protoauxin indole-3-butyric acid. Delivery of peroxisome-targeted matrix proteins depends on the PEX5 receptor docking with PEX13 at the peroxisomal membrane, and we found severely reduced import of matrix proteins and less organelle-associated PEX5 in pex13-4 seedlings. Moreover, pex13-4 physiological and molecular defects were partially ameliorated when PEX5 was overexpressed, suggesting that PEX5 docking is partially compromised in this mutant and can be improved by increasing PEX5 levels. Because previously described Arabidopsis pex13 alleles either are lethal or confer only subtle defects, the pex13-4 mutant provides valuable insight into plant peroxisome receptor docking and matrix protein import.     

Peroxisomal protein PEX13 functions in selective autophagy

PEX13 is an integral membrane protein on the peroxisome that regulates peroxisomal matrix protein import during peroxisome biogenesis. Mutations in PEX13 and other peroxin proteins are associated with Zellweger syndrome spectrum (ZSS) disorders, a subtype of peroxisome biogenesis disorder characterized by prominent neurological, hepatic, and renal abnormalities leading to neonatal death. The lack of functional peroxisomes in ZSS patients is widely accepted as the underlying cause of disease; however, our understanding of disease pathogenesis is still incomplete. Here, we demonstrate that PEX13 is required for selective autophagy of Sindbis virus (virophagy) and of damaged mitochondria (mitophagy) and that disease‐associated PEX13 mutants I326T and W313G are defective in mitophagy. The mitophagy function of PEX13 is shared with another peroxin family member PEX3, but not with two other peroxins, PEX14 and PEX19, which are required for general autophagy. Together, our results demonstrate that PEX13 is required for selective autophagy, and suggest that dysregulation of PEX13‐mediated mitophagy may contribute to ZSS pathogenesis.     

Ubiquitination of the peroxisomal targeting signal type 1 receptor, Pex5p, suggests the presence of a quality control mechanism during peroxisomal matrix protein import

PEX genes encode proteins (peroxins) that are required for the biogenesis of peroxisomes. One of these peroxins, Pex5p, is the receptor for matrix proteins with a type 1 peroxisomal targeting signal (PTS1), which shuttles newly synthesized proteins from the cytosol into the peroxisome matrix. We observed that in various Saccharomyces cerevisiae pex mutants disturbed in the early stages of PTS1 import, the steady-state levels of Pex5p are enhanced relative to wild type controls. Furthermore, we identified ubiquitinated forms of Pex5p in deletion mutants of those PEX genes that have been implicated in recycling of Pex5p from the peroxisomal membrane into the cytosol. Pex5p ubiquitination required the presence of the ubiquitin-conjugating enzyme Ubc4p and the peroxins that are required during early stages of PTS1 protein import. Finally, we provide evidence that the proteasome is involved in the turnover of Pex5p in wild type yeast cells, a process that requires Ubc4p and occurs at the peroxisomal membrane. Our data suggest that during receptor recycling a portion of Pex5p becomes ubiquitinated and degraded by the proteasome. We propose that this process represents a conserved quality control mechanism in peroxisome biogenesis.     

Genetic Interactions between PEROXIN12 and Other Peroxisome-Associated Ubiquitination Components

Most eukaryotic cells require peroxisomes, organelles housing fatty acid β-oxidation and other critical metabolic reactions. Peroxisomal matrix proteins carry peroxisome-targeting signals that are recognized by one of two receptors, PEX5 or PEX7, in the cytosol. After delivering the matrix proteins to the organelle, these receptors are removed from the peroxisomal membrane or matrix. Receptor retrotranslocation not only facilitates further rounds of matrix protein import but also prevents deleterious PEX5 retention in the membrane. Three peroxisome-associated ubiquitin-protein ligases in the Really Interesting New Gene (RING) family, PEX2, PEX10, and PEX12, facilitate PEX5 retrotranslocation. However, the detailed mechanism of receptor retrotranslocation remains unclear in plants. We identified an Arabidopsis (Arabidopsis thaliana) pex12 Glu-to-Lys missense allele that conferred severe peroxisomal defects, including impaired β-oxidation, inefficient matrix protein import, and decreased growth. We compared this pex12-1 mutant to other peroxisome-associated ubiquitination-related mutants and found that RING peroxin mutants displayed elevated PEX5 and PEX7 levels, supporting the involvement of RING peroxins in receptor ubiquitination in Arabidopsis. Also, we observed that disruption of any Arabidopsis RING peroxin led to decreased PEX10 levels, as seen in yeast and mammals. Peroxisomal defects were exacerbated in RING peroxin double mutants, suggesting distinct roles of individual RING peroxins. Finally, reducing function of the peroxisome-associated ubiquitin-conjugating enzyme PEX4 restored PEX10 levels and partially ameliorated the other molecular and physiological defects of the pex12-1 mutant. Future biochemical analyses will be needed to determine whether destabilization of the RING peroxin complex observed in pex12-1 stems from PEX4-dependent ubiquitination on the pex12-1 ectopic Lys residue.Oilseed plants obtain energy for germination and early development by utilizing stored fatty acids (Graham, 2008). This β-oxidation of fatty acids to acetyl-CoA occurs in peroxisomes, organelles that also house other important metabolic reactions, including the glyoxylate cycle, several steps in photorespiration, and phytohormone production (Hu et al., 2012). For example, indole-3-butyric acid (IBA) is β-oxidized into the active auxin indole-3-acetic acid (IAA) in peroxisomes (Zolman et al., 2000, 2007, 2008; Strader et al., 2010; Strader and Bartel, 2011). Many peroxisomal metabolic pathways generate reactive oxygen species (Inestrosa et al., 1979; Hu et al., 2012), and peroxisomes also house antioxidative enzymes, like catalase and ascorbate peroxidase, to detoxify hydrogen peroxide (Wang et al., 1999; Mhamdi et al., 2012).Peroxisomes can divide by fission or be synthesized de novo from the endoplasmic reticulum (ER). Preperoxisomes with peroxisomal membrane proteins bud from the ER and fuse, allowing matrix proteins to be imported to form mature peroxisomes (van der Zand et al., 2012; Mayerhofer, 2016). Peroxin (PEX) proteins facilitate peroxisome biogenesis and matrix protein import. Most peroxins are involved in importing proteins destined for the peroxisome matrix, which are imported after recognition of a type 1 or type 2 peroxisome-targeting signal (PTS). The PTS1 is a tripeptide located at the C terminus of most peroxisome-bound proteins (Gould et al., 1989; Chowdhary et al., 2012). The less common PTS2 is a nonapeptide usually located near the N terminus (Swinkels et al., 1991; Reumann, 2004). PTS1 proteins are recognized by PEX5 (van der Leij et al., 1993; Zolman et al., 2000), PTS2 proteins are recognized by PEX7 (Marzioch et al., 1994; Braverman et al., 1997; Woodward and Bartel, 2005), and PEX7 binds to PEX5 to allow matrix protein delivery in plants and mammals (Otera et al., 1998; Hayashi et al., 2005; Woodward and Bartel, 2005). The cargo-receptor complex docks with the membrane peroxins PEX13 and PEX14 (Urquhart et al., 2000; Otera et al., 2002; Woodward et al., 2014), and PEX5 assists cargo translocation into the peroxisomal matrix (Meinecke et al., 2010) before dissociating from its cargo (Freitas et al., 2011).After cargo delivery, PEX5 is recycled to enable further rounds of cargo recruitment (Thoms and Erdmann, 2006). This process requires a set of peroxins that is implicated in ubiquitinating PEX5 so that it can be retrotranslocated back to the cytosol. PEX5 ubiquitination is best understood in yeast. In Saccharomyces cerevisiae, Pex5 is monoubiquitinated through the action of the peroxisome-tethered ubiquitin-conjugating enzyme Pex4 and the peroxisomal ubiquitin-protein ligase Pex12 (Platta et al., 2009) and returned to the cytosol with the assistance of a peroxisome-tethered ATPase complex containing Pex1 and Pex6 (Grimm et al., 2012). S. cerevisiae Pex5 also can be polyubiquitinated and targeted for proteasomal degradation (Kiel et al., 2005). The cytosolic ubiquitin-conjugating enzyme Ubc4 cooperates with the peroxisomal ubiquitin-protein ligase Pex2 to polyubiquitinate Pex5 (Platta et al., 2009). Pex10 has ubiquitin-protein ligase activity (Williams et al., 2008; Platta et al., 2009; El Magraoui et al., 2012), but whether Pex10 directly ubiquitinates Pex5 is controversial. Pex10 promotes Ubc4-dependent Pex5 polyubiquitination when Pex4 is absent (Williams et al., 2008); however, Pex10 is not essential for Pex5 mono- or polyubiquitination (Platta et al., 2009), but rather enhances both Pex4/Pex12- and Ubc4/Pex2-mediated ubiquitination (El Magraoui et al., 2012). Recycling of the PTS2 receptor PEX7 is less understood, although the Pex5 recycling pathways are implicated in shuttling and degrading Pex7 in Pichia pastoris (Hagstrom et al., 2014).Although PEX5 ubiquitination has not been directly demonstrated in plants, the implicated peroxins are conserved in Arabidopsis, and several have been connected to PEX5 retrotranslocation. The PEX4 ubiquitin-conjugating enzyme binds to PEX22, which is predicted to be a peroxisomal membrane protein based on ability to restore peroxisome function to yeast mutants (Zolman et al., 2005). The pex4-1 mutant displays increased membrane-associated PEX5 (Ratzel et al., 2011; Kao and Bartel, 2015), suggesting that ubiquitin supplied by PEX4 promotes PEX5 retrotranslocation. PEX1 and PEX6 are members of the ATPases associated with diverse cellular activities (AAA) family and are tethered to peroxisomes by the peroxisomal membrane protein PEX26 (Goto et al., 2011; Li et al., 2014). The pex6-1 mutant displays PTS1 import defects and decreased PEX5 levels (Zolman and Bartel, 2004), suggesting that impaired PEX5 recycling can lead to increased PEX5 degradation. Indeed, pex4-1 restores PEX5 levels in the pex6-1 mutant (Ratzel et al., 2011), suggesting that Arabidopsis PEX4 also is involved in PEX5 ubiquitination and degradation when retrotranslocation is impeded.In addition to allowing for further rounds of PTS1 cargo import, several lines of evidence suggest that in the absence of efficient retrotranslocation, PEX5 retention in the peroxisomal membrane impairs peroxisome function. Slightly reducing levels of the PEX13 docking peroxin ameliorates the physiological defects of pex4-1 without restoring matrix protein import (Ratzel et al., 2011), presumably because decreasing PEX5 docking reduces its accumulation in the peroxisomal membrane. In addition, overexpressing PEX5 exacerbates rather than ameliorates the peroxisomal defects of pex4-1 (Kao and Bartel, 2015), suggesting that pex4-1 defects are linked to excessive PEX5 lingering in the peroxisome membrane rather than a lack of PEX5 available for import.The three Really Interesting New Gene (RING) peroxins (PEX2, PEX10, and PEX12) from Arabidopsis each possesses in vitro ubiquitin-protein ligase activity (Kaur et al., 2013). Null mutations in the RING peroxin genes confer embryo lethality in Arabidopsis (Hu et al., 2002; Schumann et al., 2003; Sparkes et al., 2003; Fan et al., 2005; Prestele et al., 2010), necessitating other approaches to study the in vivo functions of these peroxins. Expressing RING peroxins with mutations in the C-terminal zinc-binding RING domains (ΔZn) confers matrix protein import defects for PEX2-ΔZn and photorespiration defects for PEX10-ΔZn but no apparent defects for PEX12-ΔZn (Prestele et al., 2010). Targeting individual RING peroxins using RNAi confers β-oxidation deficiencies and impairs PTS1 cargo import (Fan et al., 2005; Nito et al., 2007). A screen for delayed matrix protein degradation (Burkhart et al., 2013) uncovered a missense pex2-1 mutant and a splicing pex10-2 mutant that both display PTS1 import defects (Burkhart et al., 2014), suggesting roles in regulating the PTS1 receptor, PEX5. A missense pex12 mutant (aberrant peroxisome morphology 4, apm4) has defects in β-oxidation and PTS1 import and increased membrane-associated PEX5 (Mano et al., 2006). These findings highlight the essential roles of the RING peroxins in Arabidopsis development and peroxisomal functions, but the RING peroxin interactions and the individual roles of the RING peroxins in PEX5 retrotranslocation remain incompletely understood.In this study, we describe a missense pex12-1 mutant recovered from a forward genetic screen for β-oxidation deficient mutants. The pex12-1 mutant displayed severe peroxisomal defects, including reduced growth, β-oxidation deficiencies, matrix protein import defects, and inefficient processing of PTS2 proteins. Comparing single and double mutants with impaired RING peroxins revealed that each RING peroxin contributes to complex stability and influences PEX5 accumulation. Furthermore, decreasing PEX4 function ameliorated pex12-1 defects, suggesting that the Glu-to-Lys substitution in pex12-1 lures ubiquitination, perhaps by pex12-1 itself, leading to PEX4-dependent degradation of the mutant protein.     

A comparative study of peroxisomal structures in Hansenula polymorpha pex mutants

In a recent study, we performed a systematic genome analysis for the conservation of genes involved in peroxisome biogenesis (PEX genes) in various fungi. We have now performed a systematic study of the morphology of peroxisome remnants ('ghosts') in Hansenula polymorpha pex mutants (pex1-pex20) and the level of peroxins and matrix proteins in these strains. To this end, all available H. polymorpha pex strains were grown under identical cultivation conditions in glucose-limited chemostat cultures and analyzed in detail. The H. polymorpha pex mutants could be categorized into four distinct groups, namely pex mutants containing: (1) virtually normal peroxisomal structures (pex7, pex17, pex20); (2) small peroxisomal membrane structures with a distinct lumen (pex2, pex4, pex5, pex10, pex12, pex14); (3) multilayered membrane structures lacking apparent matrix protein content (pex1, pex6, pex8, pex13); and (4) no peroxisomal structures (pex3, pex19).     

The E3 ubiquitin ligase SP1‐like 1 plays a positive role in peroxisome biogenesis in Arabidopsis

Peroxisomes are dynamic organelles crucial for a variety of metabolic processes during the development of eukaryotic organisms, and are functionally linked to other subcellular organelles, such as mitochondria and chloroplasts. Peroxisomal matrix proteins are imported by peroxins (PEX proteins), yet the modulation of peroxin functions is poorly understood. We previously reported that, besides its known function in chloroplast protein import, the Arabidopsis E3 ubiquitin ligase SP1 (suppressor of ppi1 locus1) also targets to peroxisomes and mitochondria, and promotes the destabilization of the peroxisomal receptor–cargo docking complex components PEX13 and PEX14. Here we present evidence that in Arabidopsis, SP1's closest homolog SP1‐like 1 (SPL1) plays an opposite role to SP1 in peroxisomes. In contrast to sp1, loss‐of‐function of SPL1 led to reduced peroxisomal β‐oxidation activity, and enhanced the physiological and growth defects of pex14 and pex13 mutants. Transient co‐expression of SPL1 and SP1 promoted each other's destabilization. SPL1 reduced the ability of SP1 to induce PEX13 turnover, and it is the N‐terminus of SP1 and SPL1 that determines whether the protein is able to promote PEX13 turnover. Finally, SPL1 showed prevalent targeting to mitochondria, but rather weak and partial localization to peroxisomes. Our data suggest that these two members of the same E3 protein family utilize distinct mechanisms to modulate peroxisome biogenesis, where SPL1 reduces the function of SP1. Plants and possibly other higher eukaryotes may employ this small family of E3 enzymes to differentially modulate the dynamics of several organelles essential to energy metabolism via the ubiquitin‐proteasome system.     

The peroxisome RING-finger complex is required for meiocyte formation in the fungus Podospora anserina

Peroxisomes are involved in a variety of metabolic pathways and developmental processes. In the filamentous fungus Podospora anserina, absence of different peroxins implicated in peroxisome matrix protein import leads to different developmental defects. Lack of the RING-finger complex peroxin PEX2 blocks sexual development at the dikaryotic stage, while in absence of both receptors, PEX5 and PEX7, karyogamy and meiosis can proceed and sexual spores are formed. This suggests a complex role for PEX2 that prompted us to study the developmental involvement of the RING-finger complex. We show that, like PEX2, the two other proteins of the complex, PEX10 and PEX12, are equally implicated in peroxisome biogenesis and that absence of each or all these proteins lead to the same developmental defect. Moreover, we demonstrate that peroxisome localization of PEX2 is not drastically affected in the absence of PEX10 and PEX12 and that the upregulation of these latter RING-finger peroxins does not compensate for the lack of a second one, suggesting that the three proteins work together in development but independent of their function in peroxisome biogenesis.     

Multiple PEX genes are required for proper subcellular distribution and stability of Pex5p, the PTS1 receptor: evidence that PTS1 protein import is mediated by a cycling receptor

PEX5 encodes the type-1 peroxisomal targeting signal (PTS1) receptor, one of at least 15 peroxins required for peroxisome biogenesis. Pex5p has a bimodal distribution within the cell, mostly cytosolic with a small amount bound to peroxisomes. This distribution indicates that Pex5p may function as a cycling receptor, a mode of action likely to require interaction with additional peroxins. Loss of peroxins required for protein translocation into the peroxisome (PEX2 or PEX12) resulted in accumulation of Pex5p at docking sites on the peroxisome surface. Pex5p also accumulated on peroxisomes in normal cells under conditions which inhibit protein translocation into peroxisomes (low temperature or ATP depletion), returned to the cytoplasm when translocation was restored, and reaccumulated on peroxisomes when translocation was again inhibited. Translocation inhibiting conditions did not result in Pex5p redistribution in cells that lack detectable peroxisomes. Thus, it appears that Pex5p can cycle repeatedly between the cytoplasm and peroxisome. Altered activity of the peroxin defective in CG7 cells leads to accumulation of Pex5p within the peroxisome, indicating that Pex5p may actually enter the peroxisome lumen at one point in its cycle. In addition, we found that the PTS1 receptor was extremely unstable in the peroxin-deficient CG1, CG4, and CG8 cells. Altered distribution or stability of the PTS1 receptor in all cells with a defect in PTS1 protein import implies that the genes mutated in these cell lines encode proteins with a direct role in peroxisomal protein import.     

PEX16 contributions to peroxisome import and metabolism revealed by viable Arabidopsis pex16 mutants

Peroxisomes rely on peroxins(PEX proteins) for biogenesis, importing membrane and matrix proteins, and fission. PEX_16, which is implicated in peroxisomal membrane protein targeting and forming nascent peroxisomes from the endoplasmic reticulum(ER), is unusual among peroxins because it is inserted co-translationally into the ER and localizes to both ER and peroxisomal membranes. PEX_16 mutations in humans, yeast, and plants confer some common peroxisomal defects; however, apparent functional differences have impeded the development of a unified model for PEX_16 action. The only reported pex_16 mutant in plants, the Arabidopsis shrunken seed1 mutant, is inviable,complicating analysis of PEX_16 function after embryogenesis. Here, we characterized two viable Arabidopsis pex_16 alleles that accumulate negligible PEX_16 protein levels. Both mutants displayed impaired peroxisome function-slowed consumption of stored oil bodies, decreased import of matrix proteins, and increased peroxisome size. Moreover,one pex_16 allele exhibited reduced growth that could be alleviated by an external fixed carbon source, decreased responsiveness to peroxisomally processed hormone precursors, and worsened or improved peroxisome function in combination with other pex mutants. Because the mutations impact different regions of the PEX_16 gene, these viable pex_16 alleles allow assessment of the importance of Arabidopsis PEX_16 and its functional domains.     

Interdependence of the Peroxisome-targeting Receptors in Arabidopsis thaliana: PEX7 Facilitates PEX5 Accumulation and Import of PTS1 Cargo into Peroxisomes

Peroxisomes compartmentalize certain metabolic reactions critical to plant and animal development. The import of proteins from the cytosol into the organelle matrix depends on more than a dozen peroxin (PEX) proteins, with PEX5 and PEX7 serving as receptors that shuttle proteins bearing one of two peroxisome-targeting signals (PTSs) into the organelle. PEX5 is the PTS1 receptor; PEX7 is the PTS2 receptor. In plants and mammals, PEX7 depends on PEX5 binding to deliver PTS2 cargo into the peroxisome. In this study, we characterized a pex7 missense mutation, pex7-2, that disrupts both PEX7 cargo binding and PEX7-PEX5 interactions in yeast, as well as PEX7 protein accumulation in plants. We examined localization of peroxisomally targeted green fluorescent protein derivatives in light-grown pex7 mutants and observed not only the expected defects in PTS2 protein import but also defects in PTS1 import. These PTS1 import defects were accompanied by reduced PEX5 accumulation in light-grown pex7 seedlings. Our data suggest that PEX5 and PTS1 import depend on the PTS2 receptor PEX7 in Arabidopsis and that the environment may influence this dependence. These data advance our understanding of the biogenesis of these essential organelles and provide a possible rationale for the retention of the PTS2 pathway in some organisms.     

Peroxisome biogenesis: advances and conundrums

Investigations of peroxisome biogenesis in diverse organisms reveal new details of this unique process and its evolutionary conservation. Interactions among soluble receptors and the membrane peroxins that catalyze protein translocation are being mapped. Ubiquitination is observed. A receptor enters the organelle carrying folded cargo and recycles back to the cytosol. Tiny peroxisome remnants - vesicles and tubules - are discovered in pex3 mutants that lack the organelle. When the mutant is transfected with a good PEX3 gene, these protoperoxisomes acquire additional membrane peroxins and then import the matrix enzymes to reform peroxisomes. Thus, de novo formation need not be postulated. Dynamic imaging of yeast reveals dynamin-dependent peroxisome division and regulated actin-dependent segregation of the organelle before cell division. These results are consistent with biogenesis by growth and division of pre-existing peroxisomes.     

Disparate peroxisome‐related defects in Arabidopsis pex6 and pex26 mutants link peroxisomal retrotranslocation and oil body utilization

Catabolism of fatty acids stored in oil bodies is essential for seed germination and seedling development in Arabidopsis. This fatty acid breakdown occurs in peroxisomes, organelles that sequester oxidative reactions. Import of peroxisomal enzymes is facilitated by peroxins including PEX5, a receptor that delivers cargo proteins from the cytosol to the peroxisomal matrix. After cargo delivery, a complex of the PEX1 and PEX6 ATPases and the PEX26 tail‐anchored membrane protein removes ubiquitinated PEX5 from the peroxisomal membrane. We identified Arabidopsis pex6 and pex26 mutants by screening for inefficient seedling β‐oxidation phenotypes. The mutants displayed distinct defects in growth, response to a peroxisomally metabolized auxin precursor, and peroxisomal protein import. The low PEX5 levels in these mutants were increased by treatment with a proteasome inhibitor or by combining pex26 with peroxisome‐associated ubiquitination machinery mutants, suggesting that ubiquitinated PEX5 is degraded by the proteasome when the function of PEX6 or PEX26 is reduced. Combining pex26 with mutations that increase PEX5 levels either worsened or improved pex26 physiological and molecular defects, depending on the introduced lesion. Moreover, elevating PEX5 levels via a 35S:PEX5 transgene exacerbated pex26 defects and ameliorated the defects of only a subset of pex6 alleles, implying that decreased PEX5 is not the sole molecular deficiency in these mutants. We found peroxisomes clustered around persisting oil bodies in pex6 and pex26 seedlings, suggesting a role for peroxisomal retrotranslocation machinery in oil body utilization. The disparate phenotypes of these pex alleles may reflect unanticipated functions of the peroxisomal ATPase complex.     

The Arabidopsis pex12 and pex13 mutants are defective in both PTS1- and PTS2-dependent protein transport to peroxisomes

Peroxisome biogenesis requires various complex processes including organelle division, enlargement and protein transport. We have been studying a number of Arabidopsis apm mutants that display aberrant peroxisome morphology. Two of these mutants, apm2 and apm4, showed green fluorescent protein fluorescence in the cytosol as well as in peroxisomes, indicating a decrease of efficiency of peroxisome targeting signal 1 (PTS1)-dependent protein transport to peroxisomes. Interestingly, both mutants were defective in PTS2-dependent protein transport. Plant growth was more inhibited in apm4 than apm2 mutants, apparently because protein transport was more severely decreased in apm4 than in apm2 mutants. APM2 and APM4 were found to encode proteins homologous to the peroxins PEX13 and PEX12, respectively, which are thought to be involved in transporting matrix proteins into peroxisomes in yeasts and mammals. We show that APM2/PEX13 and APM4/PEX12 are localized on peroxisomal membranes, and that APM2/PEX13 interacts with PEX7, a cytosolic PTS2 receptor. Additionally, a PTS1 receptor, PEX5, was found to stall on peroxisomal membranes in both mutants, suggesting that PEX12 and PEX13 are components that are involved in protein transport on peroxisomal membranes in higher plants. Proteins homologous to PEX12 and PEX13 have previously been found in Arabidopsis but it is not known whether they are involved in protein transport to peroxisomes. Our findings reveal that APM2/PEX13 and APM4/PEX12 are responsible for matrix protein import to peroxisomes in planta.